Development of Dependence in Smokers and Rodents With Voluntary Nicotine Intake: Similarities and Differences

Abstract

Introduction

Smoking and vaping throughout adolescence and early adulthood lead to nicotine dependence. Nicotine withdrawal is associated with somatic and affective withdrawal symptoms that contribute to smoking and relapse. Affective nicotine withdrawal symptoms in humans include craving for cigarettes, depression, anxiety, trouble sleeping, and cognitive deficits.

Methods

Herein, we review clinical studies that investigated nicotine dependence in people who smoke or vape. We also discuss studies that investigated the development of dependence in animals with oral nicotine intake, nicotine aerosol self-administration, and intravenous nicotine self-administration.

Results

Clinical studies report that adolescents who smoke daily develop nicotine dependence before those who smoke infrequently, but ultimately all smokers become dependent in adulthood. Preclinical studies indicate that rats that self-administer nicotine also become dependent. Rats that self-administer nicotine display somatic withdrawal signs and affective withdrawal signs, including increased anxiety and depressive-like behavior, cognitive deficits, and allodynia. Most nicotine withdrawal signs were observed in rodents with daily (7 days/week) or intermittent long access (23-hour) to nicotine. Clinical smoking studies report symptoms of nicotine dependence in adolescents of both sexes, but virtually all preclinical nicotine self-administration studies have been done with adult male rats.

Conclusions

The role of sex and age in the development of dependence in nicotine self-administration studies remains under-investigated. However, the role of sex and age in nicotine withdrawal has been thoroughly evaluated in studies in which nicotine was administered noncontingently. We discuss the need for volitional nicotine self-administration studies that explore the gradual development of dependence during adolescence and adulthood in rodents of both sexes.

Implications

The reviewed clinical studies investigated the development of nicotine dependence in male and female adolescent and young adult smokers and vapers. These studies indicate that most adolescent smokers and vapers gradually become nicotine dependent. Preclinical studies with rodents show that nicotine intake in widely used self-administration models also leads to dependence. However, almost all animal studies that investigated the development of nicotine dependence have been conducted with adult male rats. To better model smoking and vaping, it is important that nicotine intake in rats or mice starts during adolescence and that both sexes are included.

Introduction

Tobacco products and e-cigarettes are highly addictive and have adverse effects on human health. It has been estimated that there are about 1.3 billion smokers worldwide, and 8 million people die each year from smoking.¹ The use of tobacco products has been associated with the development of cancer, cardiovascular disease, and faster age-related cognitive decline.^2–4 In many Western countries, the use of tobacco products has greatly declined. For example, in the United States, the smoking prevalence declined from 42 percent in 1965 to 15 percent in 2015.⁵ It is expected that from 2010 to 2025, the greatest decline in smoking rates will be in the Americas (31%) and Africa (28%), a slower decline is expected in Europe (22%), and almost no decline is expected in the Western Pacific region (6%).¹ Eighty percent of tobacco users live in low and middle-income countries, and these countries may experience most of the adverse health and economic effects of smoking.⁶ Although smoking rates are improving, there has been a strong increase in the use of e-cigarettes (i.e. vaping).⁷ E-cigarettes were initially developed as a safe alternative to combustible tobacco products. However, recent work suggests that e-cigarettes also deliver harmful chemicals. E-cigarettes contain an e-liquid (solvent, nicotine, and flavors) that is heated to produce an aerosol. Toxic compounds have been detected in e-liquids, and heating the e-liquid further increases their levels.⁸

Prior nicotine dependence studies have mostly been done with animals that received nicotine noncontingently via minipumps. These studies have provided significant insight into the neuronal mechanisms underlying negative affective and somatic withdrawal signs.^9–11 Furthermore, an advantage of using the minipumps is that nicotine doses can easily be adjusted to ensure that rats of different ages and sex have equivalent blood nicotine levels.^12–14 Numerous previous reviews have described the effects of noncontingent nicotine administration on negative affective and somatic withdrawal signs in rodents.^15,16 However, more recently, there has been an interest in studying the development of nicotine dependence in rodents that self-administer nicotine. This review aims to provide insight into the development of dependence in humans who smoke or vape and in rodent nicotine self-administration studies (oral, inhalation, and intravenously). Drug abuse is a complex disorder that is driven by adaptations in brain networks that regulate emotional states, reinforcement learning, motor skills, and cognitive function. Cues associated with drug-taking acquire incentive-motivational value and can cause intense drug cravings and play a critical role in relapse.¹⁷ Furthermore, chronic drug use induces neuroadaptations in brain stress systems that contribute to depression, irritability, and anxiety during the acute and protracted withdrawal phase.¹⁸ Moreover, stressors cause cravings for cigarettes in people with a history of nicotine use and contribute to relapse.¹⁹ This review will specifically discuss the development of dependence in rodent nicotine self-administration studies. In prior studies, FDA-approved and potentially new smoking cessation drugs have been evaluated in animals that were made dependent through noncontingent nicotine administration.^10,20 Furthermore, smoking cessation treatments have been evaluated in nondependent rats with only a brief history of nicotine intake.^21,22 In contrast, by the time adult smokers seek treatment, they have a long history of smoking, and they are often highly dependent.²³ Therefore, new smoking cessation treatments should be evaluated in rats that are nicotine dependent and are highly motivated to self-administer nicotine. Different neuronal mechanisms might mediate drug intake in rodents during the acquisition phase and in dependent rodents with a long history of nicotine intake.²⁴ It has been suggested that the acute rewarding properties of nicotine play a role in the initiation of nicotine intake, but after the development of dependence negative reinforcement processes (drug intake to avoid withdrawal) also contribute to nicotine intake. Although the neurobiological mechanisms underlying acute nicotine withdrawal can be investigated in animals that received nicotine noncontingently, the role of negative reinforcement process in the maintenance of smoking and vaping can only be investigated in volitional nicotine intake models. Therefore, it is critical to develop nicotine self-administration models in which nicotine intake is driven by both positive and negative reinforcement processes.

It is well known that spontaneous and precipitated withdrawal signs can be investigated in rodents by exposing them to nicotine injections, osmotic minipumps with nicotine, drinking water with nicotine, tobacco smoke, and e-cigarette aerosol.¹⁵ However, much less is known about the development of dependence in rats that self-administer nicotine. In the first part of this review, we discuss the development of dependence in people who smoke or vape and the critical role of dependence in relapse. These clinical studies indicate that people start smoking or vaping during adolescence and gradually become nicotine-dependent. We then discuss studies that determined the development of dependence in rodents that self-administered nicotine. The great majority of nicotine dependence studies have been conducted with adult male rats that have been noncontingently exposed to nicotine via osmotic minipumps. We briefly discuss the development of dependence in rats that have been noncontingently exposed to nicotine via minipumps and the effects of the FDA-approved smoking cessation drugs varenicline (Chantix) and bupropion (Zyban) on nicotine withdrawal. We then discuss the development of nicotine dependence in nicotine self-administration models. These studies discuss the development of dependence in oral, inhalation, and intravenous nicotine self-administration models. The reviewed studies indicate that small changes in the self-administration protocols affect the development of dependence. The final section discusses whether the animal models mimic the development of nicotine dependence in smokers and vapers.

Development of Nicotine Dependence in Smokers

In dependent smokers, smoking cessation leads to a relatively mild somatic withdrawal syndrome, which may include increased heart rate, tremors, and gastrointestinal problems. In contrast, smoking cessation leads to severe affective withdrawal signs, including craving for cigarettes, irritability, anxiety, depression, difficulty concentrating, cognitive impairments, hyperphagia, restlessness, and sleep ­disruption.²⁵ The use of tobacco products during the acute withdrawal phase reduces the craving for cigarettes and reverses the decrease in cognitive abilities associated with nicotine withdrawal.²⁶ There is evidence that withdrawal symptoms ­contribute to relapse. The majority of people who try to quit smoking relapse during the first week of abstinence when the withdrawal symptoms are most severe.^27,28 An analysis of relapse curves of self-quitters and non-treatment controls showed that 49%–76% of smokers relapse within one week, 72%–85% in 1 month, and 80%–90% in 3 months. A recent study with homeless smokers also demonstrated that dependence and withdrawal symptoms play a role in relapse.²⁹ Smokers with a high level of dependence, as measured with the Fagerstrom test of nicotine dependence, were less likely to abstain from cigarettes on the quit day, were less likely to use the nicotine patch, and had a shorter duration of abstinence.

Most people start smoking during adolescence, and nicotine dependence develops gradually. The first signs of nicotine dependence might appear after several months of smoking, but it takes 1–4 years to develop the full nicotine dependence syndrome.^30,31 A study with 6th-grade students (11–13 years of age) from the Czech Republic showed that females have a faster increase in cigarette use than males. Furthermore, a study with Canadian 7th graders (13 years of age) showed a relationship between the smoking intensity trajectory and the development of dependence.³² The risk for dependence was minimal for adolescents who had a very low level of smoking and only slightly increased their smoking over the 39-month study period (only 12% became dependent; 6–15 cigarettes per month). In contrast, 95% of the smokers who had a high level of smoking at the beginning of the study and escalated their smoking became dependent (184–534 cigarettes per month). Furthermore, heavy smokers became dependent much faster than the light smokers. These findings indicate that there is a relationship between smoking intensity and the development of dependence in adolescents. Another study investigated the relationship between adolescent smoking trajectories and the level of dependence in adulthood.³³ As expected, adolescents with the highest level of smoking also had the highest level of dependence. However, in adulthood, all groups (low to a high levels of adolescent smoking) had a similar level of dependence. This finding indicates that heavy smoking during adolescence quickly leads to the development of dependence. However, even light smokers (few cigarettes per week) become nicotine dependent in adulthood.

The first signs of dependence can emerge soon after starting smoking. A large study with 7th graders (12–13 years of age) showed that 40% of the students who had smoked cigarettes reported signs of dependence.³⁴ The latency from the onset of monthly smoking to the first signs of dependence was 21 days for girls and 183 days for boys. When the smokers started to show signs of dependence, they only smoked a few cigarettes on one day of the week. The most-reported signs of dependence were intense cravings (28%) and needing a cigarette (31%). When the smokers tried to quit, they found it hard to concentrate (18%), were irritable (21%), had smoking urges (25%), felt anxious (21%), or depressed (11%). These findings indicate that even a very low level of smoking can lead to signs of dependence. Importantly, the development of dependence greatly increases the risk of smoking at a later time point (odds ratio 44). This study underscores that even a very low level of smoking can lead to signs of dependence, and the development of dependence predicts future smoking. Daily smoking is not necessary to become dependent as almost 40% of smokers are dependent before they start smoking daily (International Classification of Diseases, 10th Revision).³⁵

Development of Dependence in People Who Vape

Adolescent vapers have also been reported to become nicotine dependent.³⁶ A higher level of nicotine dependence was associated with starting vaping at a younger age, vaping more frequently, and using a higher nicotine concentration in the e-liquid.³⁶ Studies show that people who stop vaping display typical nicotine withdrawal signs such as irritability, anxiety, increased appetite, difficulty concentrating, depression, insomnia, restlessness, craving, anhedonia, and tremors.³⁷ Another study investigated the development of dependence in adolescents who used JUUL or non-JUUL e-cigarettes.³⁸ Forty percent of vapers showed signs of nicotine dependence, and 36 percent of vapers used e-cigarettes almost daily (20–30 days out of a 30-day period). Interestingly, users of the JUUL e-cigarettes were more likely to report symptoms of dependence (OR = 1.77) and to use e-cigarettes almost daily (OR = 1.43) than users of other brands of e-cigarettes (e.g. Blu, Vuse, and Suorin). Therefore, this study underscores that vaping leads to dependence in adolescents. The use of JUUL e-cigarettes is more likely to cause dependence than the use of other types of e-cigarettes. This is most likely because of the fact that JUUL e-cigarettes deliver a higher concentration of nicotine than most other e-cigarettes.³⁹

Noncontingent Nicotine Administration in Rodents

The development of nicotine dependence in rodents has been thoroughly evaluated by using minipumps with nicotine. In most studies, the minipumps delivered 3.16 mg/kg/day of nicotine base per day in rats, resulting in plasma nicotine levels of about 65 ng/ml.¹² The intracranial self-stimulation (ICSS) procedure can be used to determine the acute rewarding effects of drugs of abuse and the dysphoric-like state associated with drug withdrawal.⁴⁰ The acute administration of rewarding drugs increases the sensitivity to electrical stimuli (lowers the brain reward threshold) in the ICSS procedure, and drug withdrawal decreases the sensitivity to rewarding electrical stimuli (elevates brain reward thresholds).⁴¹ The studies with nicotine minipumps have shown that blockade of nicotinic acetylcholine receptors (nAChRs) with dihydro-β-erythroidine (DhβE) or mecamylamine elevates the brain reward thresholds of nicotine-treated rats.^42,43 Thus indicating that blockade of nAChRs in nicotine-treated rats induces a dysphoric-like state. Furthermore, removing the nicotine pumps also elevates the brain reward thresholds, which can last up to 72 hours.⁴³ The FDA-approved smoking cessation drugs varenicline and bupropion prevent the elevations in brain reward thresholds associated with spontaneous and precipitated nicotine withdrawal in rats.^10,20 Furthermore, cytisine, which has been used in Central and Eastern Europe as a smoking cessation drug, also prevents the elevations in brain reward thresholds associated with nicotine withdrawal in rats.^20,44,45

Nicotine pumps have also been used to study the effects of age and sex on nicotine withdrawal. In a recent study, we found that there are no sex differences in the elevations in brain reward thresholds associated with spontaneous nicotine withdrawal.⁴⁶ However, a low dose of mecamylamine elevated the brain reward thresholds in male rats but not in ­female rats. Furthermore, mecamylamine precipitated somatic withdrawal signs were detected in males but not in females. These findings indicate that sex differences in nicotine withdrawal can be observed after treatment with a low dose of mecamylamine. Nicotine withdrawal signs have also been compared between adolescent and adult rats.¹² Mecamylamine elevated the brain reward thresholds of nicotine-treated adult rats but not those of adolescent rats. Furthermore, adult rats treated with mecamylamine displayed more somatic withdrawal signs than adolescent rats. These findings indicate that adult rats display more severe withdrawal signs than adolescent rats. In addition to this, it has been shown that the removal of the minipumps leads to an increase in anxiety-like behavior, hyperalgesia, and cognitive impairments.^47–49

Place preference procedures have also been used to determine the effects of nicotine withdrawal on dysphoric-like behavior in rats. In place conditioning procedures, rodents form an association between a specific test environment and a positive or negative emotional state.⁵⁰ After the learning phase, rodents tend to spend more time in the compartment that has been paired with a positive reinforcer and less time in the compartment that has been paired with a negative reinforcer. The administration of the nAChR antagonist mecamylamine to rats treated with nicotine via minipumps for 7 days leads to conditioned place aversion.⁵¹ This confirms that chronic treatment with nicotine leads to dependence and that precipitated nicotine withdrawal leads to a dysphoria-like state. Alkhlaif et al. showed that the removal of nicotine pumps in mice leads to a reduction in sucrose preference, which is considered a sign of anhedonia or depressive-like behavior.^52,53

Somatic withdrawal signs have been widely used to investigate the development of dependence in rats. Mecamylamine and DhβE precipitate somatic withdrawal signs in rats that have been chronically treated with nicotine via minipumps.^43,46 Furthermore, removing the nicotine pumps leads to an increase in somatic withdrawal signs.⁴³ Treatment with the smoking cessation drugs varenicline and bupropion diminishes the somatic signs associated with nicotine withdrawal in rodents.^10,49 Taken together, these studies with nicotine pumps show that continuous nicotine administration rapidly (7–14 days) leads to dependence. Precipitated and spontaneous nicotine withdrawal is associated with somatic withdrawal signs and a negative emotional state.^43,54

Voluntary Oral Nicotine Intake

It has been established that rodents develop dependence when a nicotine solution is the only source of fluid. In these studies, the animals typically have access to a solution with 200 µg/ml of nicotine. Mice that are chronically exposed to drinking water with 200 µg/ml of nicotine have similar plasma nicotine levels as smokers.⁵⁵ Furthermore, mice exposed to drinking water with a high level of nicotine develop dependence as indicated by upregulation of nicotinic acetylcholine receptors (nAChRs), somatic withdrawal signs, and depressive-like behavior.^56–58 An advantage of exposing animals to nicotine via drinking water is that they develop dependence, and plasma nicotine levels are similar to those in smokers.⁵⁵ However, a disadvantage is that the animals are forced to drink a nicotine solution which might be aversive and stressful. Furthermore, because the animals are forced to drink the solution, this model does not provide insight into the positive reinforcing properties of nicotine. Furthermore, with the one-bottle model, it cannot be investigated if voluntary nicotine intake leads to the development of dependence. Despite the fact that oral nicotine intake studies have been conducted since the 1970s, only a few studies have investigated if nicotine intake in a free-choice oral self-administration paradigm leads to dependence. Voluntary oral nicotine intake is typically investigated with the two-bottle choice procedure in which the animals have access to one bottle with a nicotine solution and one bottle with water.⁵⁹ When using this model, plasma nicotine levels are relatively low, and a long exposure period is needed to induce dependence. Nesil et al. investigated the development of nicotine dependence in high- and low-nicotine-preferring adult male and female Sprague Dawley rats (Supplementary Table S1).⁶⁰ The rats were given access to a nicotine plus saccharine solution or a saccharine solution for 6 weeks. During the first 2 weeks, the nicotine concentration was 10 mg/L, and during the following 4 weeks, the nicotine concentration was 20 mg/L. Somatic withdrawal signs were counted when the rats had access to the nicotine solution (baseline) and 16 and 40 hours after removing the nicotine solution. In the females, somatic signs were increased in both the low (40 hours) and high nicotine (16 and 40 hours) preferring female rats. In the male rats, somatic signs were not increased in the low-nicotine preferring rats, but they were increased at the 40 hour time point in the high-nicotine preferring rats. This work shows that prolonged exposure to a relatively low-nicotine solution leads to the development of nicotine dependence in adult male and female rats. It is interesting to note that the females displayed more somatic withdrawal signs than the males because when nicotine is administered noncontingently, adult male rats display more somatic withdrawal signs than females.⁴⁶ This suggests that the expression of somatic signs is affected by the route of nicotine administration and the sex of the rats.

Another study investigated the development of nicotine dependence in adult C57BL/6J male and female mice using a two-bottle choice procedure.⁶¹ The mice were given access to a nicotine solution (35 μg/ml) and water for 28 days. Somatic withdrawal signs were counted before the mice were given access to the nicotine solution and 24 hours after the nicotine solution was removed. In both the males and the females, there were twice as many somatic signs after nicotine exposure than before nicotine exposure (Supplementary Table S1). This indicates that voluntary oral nicotine intake leads to nicotine dependence in mice. Intravenous drug self-administration studies suggest that intermittent access to drugs may lead to higher levels of drug intake per session than daily access.⁶² Therefore, it has been explored if intermittent access to nicotine leads to higher levels of nicotine intake in mice in the two-bottle choice paradigm than continuous exposure and if these exposure paradigms differently affect the development of dependence.⁶³ In the first experiment, adult male and female C57BL/6J mice were given daily access to nicotine (60 μg/ml) for 6 days, and then the mice were given continuous or intermittent (every other day) access to nicotine for one week. On day 14, the nicotine solution was replaced with water, and 24 hours later, somatic withdrawal signs were recorded. The mice that had either continuous or intermittent access to nicotine had more somatic withdrawal signs than the control mice that had only access to water, but the mice that had intermittent access to nicotine displayed more somatic withdrawal signs than the mice that had continuous access to nicotine (Supplementary Table S1). In the second experiment, the mice were given continuous (30 μg/ml, 24 sessions) or intermittent (12 sessions) access to nicotine for 24 days. Following this period, all the mice had continuous access to nicotine for an additional three days. Somatic withdrawal signs and anxiety-like behavior were determined 6 and 24 hours after the cessation of nicotine intake. Both nicotine groups displayed more somatic withdrawal signs and anxiety-like behavior than the control mice that only received water. Furthermore, the mice that had intermittent access to nicotine displayed more somatic withdrawal signs at the 6 hour time point and more anxiety-like behavior at the 6 and 24 hour time point than the mice that had continuous access to nicotine (Supplementary Table S1). This finding suggests that the access schedule can affect the severity of somatic and affective withdrawal signs in oral nicotine intake studies. Overall, these findings indicate that voluntary oral nicotine intake leads to dependence in male and female rodents.

Intravenous Nicotine Self-administration

Rat intravenous nicotine self-administration procedures were developed in the 1970s and early 1980s.^64,65 Studies by Corrigall and Coen showed that rats readily acquire nicotine self-administration on a limited-access (1 hour/day), fixed-ratio five, self-administration schedule after a brief period of food training.⁶⁶ In the intravenous self-administration studies, rats typically self-administer 0.03 mg/kg/inf of nicotine.^21,66 However, rats also self-administer lower and higher doses of nicotine.⁶⁷ Lower doses of nicotine lead to a higher level of operant responding but to a lower level of nicotine intake compared to the 0.03 mg/kg/inf dose. Higher doses lead to a lower level of operant responding but a higher level of nicotine intake. A PubMed search reveals that hundreds of rat nicotine self-administration have been conducted since the early 1970s, but only a few studies have investigated the development of dependence in rats that self-administered nicotine. This might be because long periods of nicotine self-administration are needed to induce dependence, and intravenous catheters have high failure rates after several weeks of drug self-administration.⁶⁸ However, improvements in catheter systems and lock solutions may allow for much longer self-administration studies.

During the early period of nicotine self-administration research, most studies were done with animals that had short access (1 hour/day, 5 days per week) to nicotine under fixed-ratio schedules of reinforcement.^21,69 Paterson and Markou investigated if these exposure conditions and also more extended access conditions lead to nicotine dependence in adult male Wistar rats.⁷⁰ They investigated ­spontaneously and precipitated somatic withdrawal signs in rats that self-administered 0.03 mg/kg/inf of nicotine for 1 hour/day 5 days/week, 1 hour/day 7 days/week, or 6 hours/day 7 days/week (Supplementary Table S2). Spontaneous somatic withdrawal signs were determined 17 hours after the 25th nicotine self-administration session. Spontaneous somatic signs were increased in rats that had self-administered nicotine for 1 hour and 6 hours/day 7 days per week but not in rats that self-administered nicotine 1 hour/day 5 days per week. Interestingly, rats that had access to nicotine for 1 and 6 hours/day 7 days per week had about the same amount of somatic signs (Supplementary Table S2). Mecamylamine precipitated withdrawal signs were investigated 17 hours after the 31st nicotine self-administration session and from 7 to 42 days after completing the nicotine self-administration sessions. Mecamylamine-precipitated somatic withdrawal signs were observed only in daily access groups (1 and 6 hours/day 7 days per week) 17 hours after nicotine self-administration. Both these groups had the same number of mecamylamine-precipitated somatic signs. Furthermore, mecamylamine-precipitated withdrawal signs were detected in all nicotine self-administration groups 7 days after the last nicotine self-administration session. At later time points, precipitated somatic withdrawal signs were only detected in the rats that ­self-administered nicotine 7 days per week, and the signs could be observed for a longer period (14 vs. 28 days) in rats that self-administered nicotine 6 hours per day than in the rats that self-administered nicotine 1 hour per day (Supplementary Table S2). Therefore, the development of nicotine dependence is affected by the number of hours of access per day and the number of sessions per week. More daily access and weekly sessions lead to more severe nicotine ­dependence. In another study, it was investigated if the dose of nicotine affects the development of nicotine dependence in a long access (23/hour day) procedure in rats.⁷¹ Adult male Wistar rats were allowed to self-administer a low (0.015 mg/kg/inf), intermediate (0.03 mg/kg/inf), or a high (0.06 mg/kg/inf) dose of ­nicotine 23 hour/day for 42 consecutive days. Precipitated somatic withdrawal signs were counted immediately after the last self-administration session (Supplementary Table S2). Mecamylamine-precipitated withdrawal signs were observed in all the groups, and more somatic signs were observed in the rats that self-administered 0.06 mg/kg/inf of nicotine than in rats that self-administered 0.015 mg/kg/inf of nicotine. There was a positive correlation between nicotine intake and the number of somatic withdrawal signs. Overall, these studies indicate that daily nicotine self-administration leads to dependence and that high doses of nicotine lead to a more severe withdrawal syndrome than low doses. Furthermore, the development of dependence can be observed in rats that have daily short (1 hour) or long access (6–23 hours) to nicotine. However, the withdrawal signs are more long-lasting in long-access rats.⁷⁰

It has also been investigated if intermittent access to nicotine (0.03 mg/kg/inf) leads to a higher level of dependence than continuous access in an intravenous self-administration procedure in adult male Wistar rats.⁶² In this study, the rats were initially given access to nicotine for 1 hour per day (acquisition phase, 5–10 days) and then switched to long access sessions (21 hours per day) for 12 consecutive days. The long-access rats were further divided into a group with daily access and a group with a combination of daily and intermittent access to nicotine (45 long access sessions). The rats that were tested under the intermittent long access schedule had a higher level of nicotine intake than the rats on the daily long access schedule (Supplementary Table S2). Spontaneous (saline injection) and precipitated (mecamylamine injection) withdrawal signs were compared between the daily and intermittent long-access rats. The intermittent long-access rats displayed slightly more spontaneous and precipitated somatic withdrawal signs than the daily long-access rats (Supplementary Table S2). In contrast, in a study with nicotine minipumps, spontaneous somatic withdrawal signs were observed in rats with daily nicotine exposure (24 hour/day ×21 days), but not in rats with intermittent nicotine exposure (12 hour/day ×21 days).⁷² In another study, the development of dependence in rats that had daily short access to 0.03 mg/kg/inf of nicotine (1 hour/day) or daily long access to the same dose of nicotine (21–23 hour/day) was investigated.⁷³ In one of the experiments, the rats were treated with mecamylamine or vehicle after 6 weeks of nicotine self-administration, and somatic signs were counted. The short and long-access rats treated with mecamylamine had the same number of somatic withdrawal signs, and treatment with mecamylamine led to more somatic signs than treatment with saline (Supplementary Table S2). In another experiment, anxiety-like behavior was determined in the elevated plus-maze test (week 10) and pain thresholds in the Von Frey test (week 12 and 13) in short and long-access rats and drug-naive controls.^74,75 The long-access rats displayed more anxiety-like behavior and had lower pain thresholds (allodynia) compared to the short-access rats and the drug-naive controls (Supplementary Table S2). In another study, O’Dell and Koob studied the development of dependence in rats that self-administered different doses of nicotine under a long access schedule (23 hour/day, 4 consecutive days/week) for 8 weeks.⁷⁶ The rats self-administered one dose of nicotine per week (0.015, 0.03, 0.06, and 0.09 mg/kg/inf) for 4 weeks, then self-administered an intermediate dose of nicotine (0.03 mg/kg/inf) for 1 week, and finally high doses of nicotine (0.06 and 0.09 mg/kg/inf) for 3 weeks. Mecamylamine-precipitated somatic withdrawal signs were observed during week 8 of nicotine self-administration (0.09 mg/kg/inf)( Supplementary Table S2). Taken together, the above-discussed studies indicate that both daily short access and daily long access to nicotine cause dependence, but spontaneous affective withdrawal signs are only observed in the long access rats.

In one of our previous studies,⁷⁷ we investigated the development of dependence in rats with intermittent short or long access to 0.03 mg/kg/inf of nicotine. The development of dependence was determined with the ICSS procedure. The rats were prepared with ICSS electrodes and intravenous catheters, and then the effects of nicotine self-administration on spontaneous and precipitated withdrawal were investigated. The rats self-administered nicotine for 10 days (1 hour/day) and were then switched to an intermittent (2 days/week) short access (1 hour/day) or long access (23 hour/day) schedule. The self-administration of nicotine lowered the brain reward thresholds in the short and long-access animals, thus indicating that nicotine self-administration has rewarding effects. However, the reward thresholds were not elevated between the self-administration sessions, thus suggesting that short or long access to nicotine does not cause spontaneous nicotine withdrawal symptoms. After 5 weeks of nicotine self-administration, the rats were treated with mecamylamine to investigate the effects of precipitated withdrawal on brain reward thresholds. Mecamylamine elevated the brain reward thresholds of the short and long-access rats, but induced a larger increase in the brain reward thresholds in the long-access rats (Supplementary Table S2). These ICSS studies ­suggest that intermittent short or long access to an intermediate dose of nicotine does not lead to spontaneous affective withdrawal signs. Precipitated withdrawal signs are observed in animals that do not display spontaneous withdrawal signs and might be an early indicator of the development of dependence.

In the above-discussed studies, the animals that self-administered nicotine and important control groups that self-administered saline were not included. However, in a recent nicotine self-administration study, yoke saline controls were included.⁷⁸ To study the development of dependence, the rats that were given access to 0.03 mg/kg/inf of nicotine in daily 2-hour sessions (6 days/week, 21 days) or they received saline. At the end of the self-administration period, the effects of cessation of nicotine intake on anxiety-like behavior (day 1 and 14), locomotor activity (day 4), depressive-like behavior (day 3 and 14), and cognition (day 17) were investigated. Cessation of nicotine intake led to increased locomotor activity (day 4), depressive-like behavior in the forced swim test on day 14, and cognitive deficits in the novel object recognition task on day 17 compared to the saline controls. Cessation of nicotine intake did not affect depressive-like behavior on day 3 or anxiety-like behavior in the light/dark box test on day 1 and 14. Somatic signs were not recorded in this study, which makes it difficult to compare the outcome of this study with other dependence studies in which somatic signs were recorded. This study suggests that daily (2-hour sessions) exposure to nicotine leads to dependence. However, it should be noted that withdrawal signs were observed after the acute nicotine withdrawal phase. In another study, spontaneous somatic withdrawal signs were investigated in rats that self-administered saline or nicotine (0.03 mg/kg/inf) under a long access schedule (12 hour/day) for 14 days.⁷⁹ Slightly more somatic withdrawal signs were observed in the nicotine group than in the saline group 12 hours after the last session (Supplementary Table S2). Paterson and Markou observed spontaneous somatic withdrawal signs 17 hours after the 25th nicotine self-administration session in rats.⁷⁰ Taken together, daily nicotine self-administration sessions for 3–4 weeks are required to induce nicotine dependence in rats.

Nicotine Aerosol Self-administration and The Development of Dependence

There has been a strong increase in vaping in adolescents, and vaping leads to the development of nicotine dependence.⁸⁰ Animal models have been developed to study vaping. It has been shown that noncontingent exposure to aerosolized nicotine causes dependence in mice and rats.^81–83 Much less is known about the development of nicotine dependence in mice or rats that self-administer nicotine aerosol. Smith et al. studied the development of nicotine dependence in male and female Wistar rats that self-administered nicotine aerosol (Supplementary Table S3).⁸⁴ The rats were allowed to lever press for nicotine aerosol (0.5 mg/ml) or vehicle (polypropylene glycol/glycerol) for 3 weeks, 1 hour per day. About half the rats discriminated between the active (nicotine or vehicle) and inactive lever. In total, 14 out of 24 rats had more active than inactive lever presses in the nicotine group, and in the vehicle group, 17 out of 24 rats had more active than inactive lever presses. Interestingly, the discriminators in the aerosol vehicle group had a higher number of active lever presses than the rats in the nicotine aerosol group, which suggests that the vehicle aerosol is reinforcing. The rats in the nicotine group displayed more somatic withdrawal signs after treatment with mecamylamine than rats in the vehicle group. Furthermore, treatment with mecamylamine decreased the pain threshold (i.e. allodynia) in the nicotine rats but not in the controls. Furthermore, 3 weeks after the last nicotine exposure session, the rats displayed increased anxiety-like behavior, allodynia, and downregulation of α4 and β2 nAChR subunit mRNA levels in the nucleus accumbens. This work suggests that the nicotine aerosol is less reinforcing than vehicle aerosol and that exposure to the nicotine aerosol leads to dependence.

Duration of Nicotine Exposure in Humans and Animal Models

People often start smoking or vaping during adolescence and start trying to quit in adulthood.^85,86 It takes several attempts to quit smoking, and the average age of quitting is about 40 years of age, which indicates that many people have smoked ≥25 years before they quit.⁸⁶ People who start smoking during adolescence are more dependent when they try to quit and they are also more likely to relapse than people who start smoking as an adult.^85,87,88 In contrast, in most animal studies, the onset of nicotine self-administration is during adulthood and nicotine withdrawal is investigated after only 4 to 5 weeks of nicotine self-administration.^70,77 Intravenous nicotine self-administration procedures are currently the gold standard to determine the reinforcing properties of nicotine. However, the rapid growth of rodents during adolescence limits the use of catheters during this development period. Furthermore, catheter patency issues often prevent long-term (>2 months) nicotine self-administration studies. Nicotine intake during ­adolescence and early adulthood can be investigated through oral nicotine intake and nicotine aerosol self-administration procedures, which don’t require IV catheters.^89,90 Therefore, oral nicotine intake and nicotine aerosol self-administration procedures could provide important insight into environmental and neurobiological factors that contribute to nicotine intake in adult rats that started nicotine intake during adolescence. To model smoking, it is pivotal to develop animal models in which nicotine intake starts during adolescence and continues into adulthood or old age. It is critical to determine why adolescent onset smoking leads to a higher level of nicotine dependence and relapse rates compared to adult smoking. This difference between adolescent and adult smoking could be due to a longer nicotine exposure period, however, it might also be possible that exposure to nicotine during adolescence leads to specific neuronal changes that cause more severe dependence and a greater risk for relapse.

Nicotine Intake in Humans and Animal Models

People generally smoke and vape during the day and not at night.^91,92 Therefore, smokers have high daytime nicotine levels and low nighttime levels. Traditional nicotine self-administration and passive exposure procedures have provided initial insight into the reinforcing properties of nicotine. However, these procedures did not model the complexities of smoking very well. In most studies, the animals were only allowed to self-administer nicotine for one hour per day during the light phase (when rats are inactive) of the light/dark cycle, or nicotine was administered noncontingently via minipumps (24 hour/day) or injections.^21,43,66,93 However, in more recent studies, rats have been given long access (23 hour/day) to nicotine in daily or intermittent self-administration sessions.^62,76 Interestingly, the long-access rats self-administer more nicotine during the dark phase (when rats are active) than during the light phase.^71,77 This suggests that the circadian rhythm of nicotine intake in the long access self-administration procedures closely models the circadian rhythm of smoking in humans. Furthermore, in order to better model vaping, nicotine aerosol exposure procedures have been developed in which animals are passively exposed to the nicotine aerosol or have been trained to self-administer nicotine aerosol.^81,90,94,95

The pharmacokinetics of nicotine and the delivery rate affects the reinforcing and motivational properties of nicotine. In humans, the bioavailability of inhaled nicotine is 80%–90%.⁹⁶ A single puff on an e-cigarette or tobacco cigarette causes a rapid rise in brain nicotine levels (50% of maximum brain nicotine concentration in less than 30 seconds).^97,98 Similarly, in rodents, both intravenous nicotine self-administration and inhalation procedures model smoking well in that both intravenous nicotine intake and nicotine aerosol exposure cause a rapid rise in brain nicotine levels.^99–101 Furthermore, nicotine levels in smokers are similar to those in rodents that self-administer nicotine. Plasma nicotine levels in heavy smokers are about 40 ng/ml.^102,103 Intravenous nicotine self-administration (0.03 mg/kg) and the inhalation of nicotine aerosol (0.5 mg/ml) in rats leads to a plasma nicotine level of 60 to 80 ng/ml.^67,84,104 The oral bioavailability of a nicotine solution is very low (20 % bioavailability) because of high hepatic first-pass metabolism.⁹⁶ Because of poor bioavailability, voluntary oral nicotine intake leads to a somewhat lower plasma nicotine level (15–18 ng/ml of nicotine).¹⁰⁵ Therefore, oral nicotine intake leads to a slow increase in nicotine levels, and then the nicotine levels remain relatively stable. Because of the slow increase in nicotine levels after oral intake, oral nicotine intake is less reinforcing than intravenous nicotine self-administration. The plasma half-life (t1/2) of nicotine is 6–7 minutes in mice and about 1 hour in rats.^55,106 In humans, the plasma half-life of nicotine after smoking or an intravenous nicotine infusion is about 2 hours.⁹⁶ Because of the short half-life of nicotine, blood nicotine levels drop quickly after smoking, and low-nicotine levels lead to withdrawal signs in nicotine-dependent smokers. Therefore, smokers typically smoke at regular intervals to maintain stable blood nicotine levels and avoid withdrawal signs.¹⁰⁷ Similarly, rodent self-administration studies suggest that rodents carefully regulate their nicotine intake.^{66,77,108,109} Smoking during the night and soon after waking in the morning predicts the level of nicotine dependence.^92,110 Night smokers are more dependent than people who do not smoke during the night. Interestingly, rat nicotine self-administration studies show that nicotine intake during the light phase increases over time, and nicotine intake during the dark phase decreases.^71,77 Thus, in both human and animal models of smoking, nicotine intake during the inactive period of the light/dark cycle increases over time. In addition to this, brain imaging studies show that there is a strong overlap in brain areas that are activated by treatment with nicotine in rodents and humans.^111,112 The rewarding effects of nicotine are primarily mediated by the activation of α4β2 containing nAChRs. Human brain imaging studies show that smoking upregulates α4β2 nAChRs.^113,114 Similarly, nicotine self-administration in rodents leads to the upregulation of α4β2 nAChRs.^{67,72,115,116} This suggests that although the route of nicotine intake is different in rodents and humans, the rodent nicotine self-administration models mimic the neuroadaptive changes associated with smoking in humans.

Smoking Cessation Treatments in Humans and Animal Models

The FDA-approved smoking cessation drugs varenicline (Chantix) and bupropion (Zyban) decrease nicotine intake and nicotine withdrawal in rats.^10,20,21,117 However, many other compounds have been evaluated in animal models for smoking. In particular, compounds that decrease noradrenergic transmission have been evaluated. Prazosin, clonidine, and propranolol inhibit noradrenergic transmission by blocking α1- or β-adrenergic receptors or stimulating presynaptic α2-adrenergic receptors.¹¹⁸ The α2-adrenergic receptor agonist clonidine attenuates footshock-induced reinstatement of nicotine-seeking behavior in rats.¹¹⁹ However, clonidine does not attenuate the elevations in brain reward thresholds associated with nicotine withdrawal.¹¹⁸ A meta-analysis with seven studies and 989 patients showed that clonidine aids in smoking cessation.¹²⁰ The α1-adrenergic receptor antagonist prazosin has also been evaluated in nicotine self-administration procedures. Prazosin reduces nicotine self-administration and nicotine prime and cue-induced reinstatement of nicotine seeking in rats.^121,122 Furthermore, prazosin dose-dependently prevents the elevations in brain reward thresholds associated with nicotine withdrawal.¹¹⁸ Although animal studies provide strong evidence for a role of α1-adrenergic receptors in the reinforcing properties of nicotine, the role of α1-adrenergic receptors in smoking and relapse has not been evaluated in clinical studies. The effects of the nonselective β-adrenergic receptor antagonist propranolol on the reinstatement of nicotine-seeking and nicotine withdrawal have been determined in animal models. Propranolol decreases cue-induced reinstatement of nicotine-seeking in rats.¹²³ However, propranolol does not attenuate the elevations in brain reward thresholds associated with nicotine withdrawal.¹¹⁸ A meta-analysis with two studies and 139 patients suggests that treatment with propranolol does not affect smoking cessation.¹²⁰ Preclinical studies have provided extensive evidence for a role of corticotropin-releasing factor (CRF) in nicotine addiction. CRF type 1 (CRF1) receptor antagonists diminish the elevations in brain reward thresholds, anxiety-like behavior, and hyperalgesia associated with nicotine withdrawal in rats.^42,73 Despite solid evidence for a role of CRF in nicotine intake and relapse, there are no clinical studies that have investigated the effects of CRF antagonists on smoking and relapse. Although CRF antagonists showed promise for treating psychiatric disorders, the elevation of liver enzyme activity after treatment with the CRF1 antagonist NBI 30775 (R121919) dampened interest in the clinical development of CRF1 antagonists.¹²⁴

Discussion and Knowledge Gaps

We have reviewed clinical and preclinical studies that investigated the development of nicotine dependence. Clinical studies indicate that people start smoking and vaping at a very young age. Almost all smokers start as light smokers but smoking increases over time. Adolescent heavy smokers rapidly become nicotine dependent. However, ultimately, adolescent light and heavy smokers reach a similar level of dependence in adulthood. Much less is known about the development of dependence in e-cigarette users, but emerging ­evidence indicates that adolescent e-cigarette users also become nicotine-dependent.^80,125 The reviewed clinical studies include males and females, and sex differences in smoking trajectories and the development of dependence have been identified.^34,126 The development of dependence has also been investigated in animal studies.¹²⁷ However, most animal studies were conducted with male rats, and relatively high doses of nicotine were administered noncontingently. In contrast, humans initially chose to use nicotine products and carefully titrate their nicotine intake. Furthermore, nicotine products are used by males and females (60% of females and 45% of males tried e-cigarettes). Therefore, there is a need for studies that investigate the acquisition of nicotine intake and the development of dependence in adolescence and early adulthood. Some animal studies have investigated the development of dependence in rats that self-administer nicotine. However, these studies have been mainly conducted with adult male rodents. We are not aware of any intravenous nicotine self-administration studies that investigated the development of dependence in both males and females. Furthermore, there are no studies in which nicotine intake started during adolescence, and nicotine dependence was investigated throughout adolescence and early adulthood. Technical challenges make it difficult to study the development of dependence in adolescent rats that self-administer nicotine. First, the adolescent period is very brief in rodents (postnatal days 28–42 early adolescent and postnatal days 43–55 late adolescent/emerging adulthood), and it requires a long time for rats to develop dependence through intravenous nicotine self-administration.^128,129 Previous work by Paterson et al. suggests that it may take 30 days of daily access to develop nicotine dependence.⁷⁰ Furthermore, because of the rapid growth of the animals, catheters have a higher failure rate in adolescent rats than in adult rats.¹³⁰ These problems could potentially be circumvented by replacing the catheter. However, a surgical procedure and recovery during the study would prevent nicotine intake for about a week and may have a negative effect on nicotine intake. An alternative approach would be to allow voluntary oral nicotine intake or nicotine aerosol exposure during adolescence, followed by intravenous nicotine self-administration in adulthood. Previous work has shown that male and female adolescent rats consume nicotine (5–6 mg/kg each week) in a 2-bottle choice paradigm and adult rats self-administer nicotine aerosol.^84,89 Therefore, these oral nicotine intake and nicotine aerosol self-administration models could potentially be used to investigate the gradual development of dependence in adolescent smokers and vapers.

Oral nicotine intake and nicotine aerosol self-administration models also have some disadvantages. Smoking leads to a rapid rise in nicotine levels in the brain, which is followed by a rapid decline in nicotine levels.^97,131 Oral nicotine intake leads to a slow increase in nicotine levels, and then the nicotine levels remain relatively stable. Therefore, oral nicotine intake is not as reinforcing as smoking or intravenous nicotine self-administration. Exposing animals to nicotine ­aerosol also differs from human smoking. Rodents are obligatory nose breathers, and humans are nose/oral breathers and inhale tobacco smoke and e-cigarette aerosol through the mouth. Inhalation through the mouth leads to a higher percentage of the particles delivered to the lungs. E-cigarette aerosol particles are 1–2 µm in diameter.¹³² The respirable fraction of these particles is 90% in humans but only 30%–60% (lowest for the large particles) in mice and 30-80% in rats.¹³³ Whole-body exposure could also lead to exposure via other routes, such as the mouth and eyes. A study with a radioactive aerosol showed that in whole-body exposure sessions, almost 50% total body burden of aerosol was on the fur, and the gastrointestinal system contained more radioactive material than the lungs.¹³⁴ A total of 60%–80% of the aerosol that is deposited on the fur might be orally ingested. Therefore, oral nicotine intake may contribute to nicotine exposure in the nicotine aerosol studies. It has also been suggested that animals could diminish exposure to the aerosol by burying their nose in their fur or the corner of the cage.¹³⁵

Conclusion

In conclusion, the reviewed studies indicate that people start smoking and vaping during early adolescence, and users of nicotine products gradually become nicotine dependent. By the time people try to quit smoking or vaping, they have been using nicotine products daily for years or decades and are highly dependent. In contrast, most animal studies have been conducted with animals that have been made dependent through noncontingent administration of high doses of nicotine, or the animals only briefly self-administered nicotine and are not dependent. However, rodent nicotine self-administration protocols have been developed that lead to the development of dependence. Rodents will press a lever or nose poke for the delivery of nicotine aerosol or intravenous nicotine and orally consume nicotine in the two-bottle choice paradigm. The development of dependence is almost always confirmed by measuring precipitated or spontaneous somatic withdrawal signs. Precipitated withdrawal is indicative of adaptations in cholinergic transmission,¹³⁶ and animals that show precipitated withdrawal signs may not yet show spontaneous withdrawal signs.^77,137 Spontaneous somatic withdrawal signs might therefore be a better indicator of dependence than precipitated withdrawal signs. Furthermore, when animals display spontaneous withdrawal signs, nicotine intake might be partly driven by negative reinforcement processes. Nicotine withdrawal has also been associated with changes in locomotor activity, anxiety- and depressive-like behaviors, cognitive function, and pain thresholds, but these measures are less widely reported than somatic withdrawal signs in self-administration models.^138,139

In conclusion, there is a need for better animal models to investigate smoking and vaping and the gradual development of dependence during adolescence and young adulthood. It is also critical to include males and females because there are sex differences in nicotine intake and withdrawal. It is also important to evaluate new drug treatments in nicotine-dependent animals that have become dependent through voluntary nicotine intake. Better animal models may aid in the development of novel treatments that will help nicotine-dependent smokers and vapers quit.

Supplementary Material

A Contributorship Form detailing each author’s specific involvement with this content, as well as any supplementary data, are available online at https://academic.oup.com/ntr.

Funding

A.B. was supported by a National Institute on Drug Abuse (NIDA)/National Institutes of Health (NIH) grant DA046411.

Declaration of Interests

None declared.